AtSWEET11 and AtSWEET12 transporters function in tandem to modulate sugar flux in plants

Abstract The sugar will eventually be exported transporter (SWEET) members in Arabidopsis, AtSWEET11 and AtSWEET12 are the important sucrose efflux transporters that act synergistically to perform distinct physiological roles. These two transporters are involved in apoplasmic phloem loading, seed filling, and sugar level alteration at the site of pathogen infection. Here, we performed the structural analysis of the sucrose binding pocket of AtSWEET11 and AtSWEET12 using molecular docking followed by rigorous molecular dynamics (MD) simulations. We observed that the sucrose molecule binds inside the central cavity and in the middle of the transmembrane (TM) region of AtSWEET11 and AtSWEET12, that allows the alternate access to the sucrose molecule from either side of the membrane during transport. Both AtSWEET11 and AtSWEET12, shares the similar amino acid residues that interact with sucrose molecule. Further, to achieve more insights on the role of these two transporters in other plant species, we did the phylogenetic and the in‐silico analyses of AtSWEET11 and AtSWEET12 orthologs from 39 economically important plants. We reported the extensive information on the gene structure, protein domain and cis‐acting regulatory elements of AtSWEET11 and AtSWEET12 orthologs from different plants. The cis‐elements analysis indicates the involvement of AtSWEET11 and AtSWEET12 orthologs in plant development and also during abiotic and biotic stresses. Both in silico and in planta expression analysis indicated AtSWEET11 and AtSWEET12 are well‐expressed in the Arabidopsis leaf tissues. However, the orthologs of AtSWEET11 and AtSWEET12 showed the differential expression pattern with high or no transcript expression in the leaf tissues of different plants. Overall, these results offer the new insights into the functions and regulation of AtSWEET11 and AtSWEET12 orthologs from different plant species. This might be helpful in conducting the future studies to understand the role of these two crucial transporters in Arabidopsis and other crop plants.


| INTRODUCTION
Plant sugar will eventually be exported transporter (SWEET) proteins were initially identified in the model organism Arabidopsis thaliana (Chen et al., 2010). In total, 17 members were identified in Arabidopsis, classified into four different sub-clades (Breia et al., 2021;Chen et al., 2010;. All members of Clade III including AtSWEET11 and AtSWEET12 were characterized as sucrose efflux transporters using forster resonance energy transfer (FRET) based sucrose sensors expressed in human embryonic kidney (HEK293T) cells, yeasts, and time-dependent efflux of [ 14 C] sucrose in Xenopus oocytes (Chen et al., 2012). AtSWEETs are bidirectional transporters that facilitate the diffusion of sucrose molecules down the concentration gradient and adapt a uniporter transport mechanism as their transport activity is pH independent. Kinetic studies of AtSWEET12 (Km for sucrose uptake and efflux was $70 mM and 10 mM respectively) revealed that SWEETs are low affinity sucrose transporters (Chen et al., 2012). AtSWEET11 and AtS-WEET12 share almost 88% amino acid similarity (Chen et al., 2012) and both were shown to exhibit the substrate flexibility as they transported glucose and fructose in addition to sucrose (Le Hir et al., 2015). These SWEET proteins are heptahelical transmembrane (TM) transporters with two internal parallel triple-helix bundles (THB) that are interconnected by the nonconserved helix TM4 (Han et al., 2017;Tao et al., 2015). The two THB domains have a twofold rotation symmetry perpendicular to the membrane plane and have a characteristic 1-3-2 and 5-7-6 topological arrangement (Anjali et al., 2020;Han et al., 2017).
Among the members of AtSWEETs, AtSWEET11 and AtSWEET12 are particularly known to be expressed in all tissues, including the leaves, roots, seeds, siliques, and flowers (Chen et al., 2012).
In Arabidopsis, AtSWEET11 and AtSWEET12 participate in pivotal processes such as phloem loading (Chen et al., 2012), xylem development (Le Hir et al., 2015), and seed filling . This demonstrates that AtSWEET11 and AtSWEET12 are crucial transporters required in major developmental and physiological processes.
Besides, these two transporters optimize the sugar flux in response to varying environmental conditions. AtSWEET11 and AtSWEET12 have been shown to have role during interactions with pathogens (Chen et al., 2010;Gebauer et al., 2017;Walerowski et al., 2018).
Nevertheless, the dynamic role of these two transporters in multiple integrated aspects of plant physiology has raised considerable interest in their structure and molecular regulatory mechanisms. In this article, we probe the structural aspects of sucrose binding pockets from

| Bacterial pathogen and plant inoculations
The host bacterial pathogen of Arabidopsis, Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) were grown at 28 C with continuous shaking at 150 rpm in King's B (KB) medium (liquid) (Cat# M1544; HiMedia Laboratories) containing rifampicin at 50 μg/ml. Bacterial cultures were grown overnight (12 h) to obtain an optical density of .4 at 600 nm (OD 600 = .4). Bacterial cells were collected by centrifugation at 4,270 Â g for 10 min, washed thrice in sterile water, and re-suspended in sterile water at desired concentrations. The concentrations used for the inoculation of the leaves (32-day-old plants) were 5 Â 10 5 colony-forming units (CFU)/mL. The 5 ml of bacterial suspension was syringe-infiltrated on the abaxial surface of fully expanded leaves using a needleless syringe. The inoculated plants were maintained in a growth chamber at 20 C.

| Molecular dynamics (MD) trajectory analysis
The full-length amino acid sequences of AtSWEET11 and AtS-WEET12 were obtained from TAIR (https://www.arabidopsis.org) and submitted to psiPRED server (http://bioinf.cs.ucl.ac.uk/psipred) for sequence based secondary structure prediction. Psipred predicted transmembrane helices and cytoplasmic disordered C-terminal region for both the sequences. For homology modeling, the methodology from Sastry et al. (2013) was followed. The full length AtSWEET11 and AtSWEET12 protein sequence were submitted to the Robetta server (http://robetta.bakerlab.org) and the output models for AtS-WEET11 (PD algorithm) and AtSWEET12 (TR algorithm) with .76 and .74 confidence score respectively were selected after visualizing them.
The models selected had minimal estimated positional error for the residues. However, the C-terminal for both the predicted proteins was highly disordered with maximal regions modeled as loops. The 3D coordinates of AtSWEET11 and AtSWEET12 homology models were prepared using the protein preparation wizard of the Schrodinger suite. The missing residues and side chains were filled and bond order errors were corrected, followed by H-bond optimisation and a restrained minimisation with a cut-off of .3 Å. The prepared protein models were simulated to equilibrate in a POPC lipid bilayer at 300 K.
POPC lipid was automatically placed perpendicular to the transmembrane helices and the systems were solvated using TIP3P water model. The MD system was electrostatically neutralized by placing counter Cl À ions and .15 M KCl was added to provide adequate ionic strength. The prepared systems were relaxed by default desmond relaxation protocols before being simulated for 600 ns with a recording interval of 50 ps. The C-terminal domain was excluded due to high level of disorder and only the stable transmembrane domain of 1-219 amino acids was used for docking and MD analysis.

| RMSD plot analysis
The 3D coordinates of sucrose were extracted from PDB:3LDK and possible conformers were generated using Ligprep. The 3D coordinates of the AtSWEET11 and AtSWEET12 transmembrane domain were extracted from the last frame of the 600 ns MD and subjected to a sitemap analysis. The best scoring sites with site scores of 1.139 and 1.134 were predicted at the central cavity for AtSWEET11 and AtSWEET12 respectively. These sites were selected to generate receptor grids for docking sucrose. The sucrose was docked at the prepared grid using the XP (Extra precision) docking mode using Glide.
Output poses were visualized for interactions and clashes, and binding energy calculations were calculated through Prime-MMGBSA module of Schrodinger suite to select the best pose for MD run. The sucrose docked AtSWEET11 and AtSWEET12 were prepared in lipid bilayer membrane environment and simulated for 500 ns and 1us respectively to study their interaction dynamics. Thermal MM-GBSA script was ran on the MD trajectory of AtSWEET11 and AtSWEET12 to obtain dG binding score w.r.t. Frames. The most stable complex according to the dG binding score was exported to study interactions/ representation.

| Identification of AtSWEET11 and AtSWEET12 orthologous proteins
The potential orthologs of AtSWEET11 and AtSWEET12 were identified by comparing the protein sequence against the proteomes of 39 economically important plant species from 20 different families.
SWEET protein sequences were retrieved from EnsemblPlants database (http://plants.ensembl.org/). The best five hits for the orthologs of each plant species were again compared against the Arabidopsis proteome. The best three hits were then examined for the presence of MtN3_slv domain (IPR018179) using Pfam database (http://pfam. xfam.org/) and Conserved Domain Database (CDD) (http://www.ncbi. nlm.nih.gov/Structure/cdd/wrpsb.cgi) and also checked for the presence of transmembrane helices (TMHs) in protein sequences using TMHMM Server v.2.0 (http://www.cbs.dtu.dk/services/TMHMM/) with default parameters. Finally, the first best hit obtained after passing through these steps was considered as a potential orthologs of AtSWEET11 and AtSWEET12 for each plant species.

| Time-tree and phylogenetic analysis
The time-tree analysis of thirty-nine different plant species from twenty different families was performed. The species names were taken as input and time-tree was generated using the MEGA X (www.  (Kumar et al., 2018;Tamura et al., 2012). The evolutionary analysis was conducted in MEGA X.

| Gene structure, chromosome localization and cis-elements analysis
The gene structure analysis including exon-intron arrangement were conducted for the orthologs of AtSWEET11 gene and AtSWEET12 gene from thirty-nine different plant species using Gene Structure Display Server 2.0 (http://gsds.gao-lab.org/index.php). The chromosome location of the orthologous genes was identified using EnsemblPlants database (http://plants.ensembl.org/). The ciselements were identified in 1.5 kb 5 0 upstream promoter regions of AtSWEET11 and AtSWEET12 orthologs from 39 different plant species using PlantCARE (http://bioinformatics.psb.ugent.be/ webtools/plantcare/html/).

| Protein motif analysis and tertiary structure prediction
The conserved motifs in orthologous proteins for AtSWEET11 and AtSWEET12 were identified using MEME_suite (https://meme-suite. org/meme/). The value of 0 or 1 was used for a specific motif and 20 was set as the upper limit of motifs. The motif length was set at 6-50 amino acids. All motifs were then annotated using InterProScan database (http://www.ebi.ac.uk/Tools/pfa/iprscan/). The tertiary structures of the proteins orthologus to AtSWEET11 and AtSWEET12 were predicted using Robetta (https://robetta.bakerlab.org/).

| RT-qPCR analysis
The total RNA was extracted from the leaf samples using TriZol from TM2, P150 from TM5, and P167 from TM6) plays a major role in alternating access mechanism of sucrose transport by inducing concerted structural arrangements in the TM helices ( Figure 1h).
Replacing any one of these prolines with alanine in AtSWEET1 abolishes the transport activity (Tao et al., 2015). When adjacent residues to these conserved prolines were mutated to alanine in AtSWEET1, it resulted in reduced glucose transport (Tao et al., 2015), indicating the importance of these residues in the transport cycle.
The other conserved residues that form the putative intracellular gate are shown in Figure 1h. When co-expressed with a wild type transporter, mutation of V188A in AtSWEET1 in the extrafacial gate showed complete transport inhibition while P23T in the cytosolic gate played an allosteric role, both displaying a dominant-negative effect in substrate transport (Han et al., 2017;Tao et al., 2015;Xuan et al., 2013).

| Analysis of AtSWEET11 and AtSWEET12 orthologs from different plant species
The potential orthologs of AtSWEET11 and AtSWEET12 were identified by comparing the protein sequence against the proteomes  were represented in the form of heat map. Color bar ranging from dark red to white indicate the levels of transcript expression from high to low or undetected respectively. (d), the transcript expression pattern of AtSWEET11 and AtSWEET12 genes after Pst DC3000 inoculations is shown here. The 32-d-old Arabidopsis wild-type plants were syringe-inoculated with sterile water (mock), Pst DC3000 at 5 X 10 5 CFU/ml. samples were collected at 0 and 16 hpi. The transcript levels were measured by RT-qPCR. For each treatment, the fold change in gene expression levels were calculated over mock-treated wild-type samples and were expressed as log2 values. Bars represent the transcript expression pattern of genes. Bars above and below the horizontal axis indicate the upregulation and downregulation in transcript expression respectively. Asterisks indicate significant difference from mock-treated wild-type (student's t test; *P < .01). Data were obtained from mean of three biological replicates (n = 3) and error bars show ± standard error of mean. (e), the transcript levels of AtPR1 and AtPR5 the defense responsive genes in atsweet11 and atsweet12 mutants. Leaf samples were collected from 32-d-old Arabidopsis wild-type and mutant plants. The transcript levels were measured by RT-qPCR. The fold change in expression levels were obtained over wild-type and expressed as Log 2 values. Bars represent the transcript expression pattern of AtPR1 and AtPR5 genes. Bars above and below the horizontal axis indicate the up-regulation and down-regulation in transcript expression respectively. Asterisks (*) indicate a significant difference from wild-type (student's t test; *P < .01). Data were obtained from mean of four biological replicates (n = 4) and error bars show ± standard error of mean.
available for the orthologs of only a few species (Supplementary File 1 f, g, h). against a concentration gradient (Ayre, 2011;Giaquinta, 1983;Srivastava et al., 2008;Srivastava, Dasgupta, et al., 2009;Srivastava, Ganesan, et al., 2009). Chen et al. (2012) showed that plants carrying mutations in both AtSWEET11 and AtSWEET12 showed moderate growth defects and accumulated excessive sugar in the leaves due to blockage in the sugar translocation pathway. However, single mutants of either of these genes did not affect the plant (Chen et al., 2012), implying a redundant function of AtSWEET11 and AtSWEET12 in sucrose transport during phloem-loading process (Figure 8a). AtS-WEET11 and AtSWEET12 are also involved in long-distance translocation of sucrose in both the source and sink of plants grown in vitro ( Figure 8b) (Papaioannou, 2018). The study suggests that the directionality of sucrose transport by AtSWEET11 and AtSWEET12 can be reversed in accordance to the sucrose concentration gradient.

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Besides, the study showed that the transgenic with the single mutant atsweet12 and AtSWEET11 overexpression translocated more sucrose from the source (root tissues) to the sink (leaf tissues). However, this was not observed in the reverse case, i.e., atsweet11 mutant with AtSWEET12 overexpression (Papaioannou, 2018). In other words, in the absence of AtSWEET12, sucrose transport is overtaken by Pseudomonas simiae WCS417r, and the beneficial effect of P. simiae was lost in Arabidopsis atsweet11;12 double mutants (Desrut et al., 2020). This suggests that AtSWEET11 and AtSWEET12 could possibly function in controlling the sugar supply from the shoot to the root and its distribution to the PGPR, which might positively impact the plant-PGPR interaction (Figure 8g). Similarly, in other study AtSWEET12 was shown to suppress multiplication of different species of P. syringae by restricting sucrose availability to these foliar bacterial pathogens in the leaf apoplast. The study traced the AtSWEET11-mediated sucrose flux to be modulated through AtS-WEET12 via plasma membrane targeting and an oligomerizationdependent regulatory mechanism in Arabidopsis. This also indicates the exclusive role of AtSWEET12 in suppressing bacterial multiplication and the role of AtSWEET11 in supplying sugars to bacterial pathogens in the apoplast (Fatima & Senthil-Kumar, 2021 Figure 4). Nevertheless, future studies are required to validate the role of AtSWEET11 and F I G U R E 8 AtSWEET11 and AtSWEET12 localization, involvement in the sucrose transport pathway, and related physiological functions and stress responses in Arabidopsis. AtSWEET11 and AtSWEET12 transporters are reported to play a major role in the roots, hypocotyl, leaf/stem (tracheid), and seeds. (a), during apoplasmic phloem loading, phloem parenchyma-localized AtSWEET11 and AtSWEET12 transport sucrose from the mesophyll cells to the phloem apoplast. next, AtSUC2 transports the sucrose from the apoplast to the sieve element-companion cell complex (Chen et al., 2012). (b), in in vitro grown plants with exogenous carbon supply, the roots act as a source tissue, and the leaves become the sink. The directionality of sucrose transport by AtSWEET11 and AtSWEET12 gets reversed according to the sucrose concentration gradient, i.e., from the root (source) to the leaf tissues (sink) (Papaioannou, 2018). (c), during xylem development, AtSWEET11 and AtSWEET12 are localized in xylem parenchyma cells in the inflorescence stem and facilitate secondary cell wall formation by delivering carbon sources to the developing xylem cells in the inflorescence (Le Hir et al., 2015). (d), during seed filling, AtSWEET15 localized in the outer integument transports sucrose into the apoplast. AtSWEET11 localized in the inner integument facilitates sucrose supply to the developing embryo from the micropylar endosperm. AtSWEET12 localized in the micropylar end of the seed coat facilitates sucrose transport to the seed coat region. Together, these three transporters, among others, are involved in sink-drawing ability during seed development/grain filling . (e), during water deficit conditions, AtSWEET11, AtSWEET12, and AtSUC2 are involved in drawing more sucrose to the root cells. Under water stress, these transporters accumulate in the roots and unload sucrose from the apoplast to the sink cells in the roots and facilitate root growth by allocating more sucrose from the leaves to the roots (Durand et al., 2016). (f), AtSWEET11 and AtSWEET12 facilitate sugar delivery towards the pathogen (here, Plasmodiophora brassicae at the site of infection in the hypocotyl region) (Walerowski et al., 2018). (g), AtSWEET11 and AtSWEET12 participate in phloem unloading of sucrose, especially to the lateral roots. These transporters control the sugar supply from the shoot to the root and then distribute sugars to plant growth-promoting rhizobacteria (PGPR, i.e., here, Pseudomonas simiae WCS417r in the rhizosphere region) (Desrut et al., 2020).

| CONCLUSION AND FUTURE DIRECTIONS
AtSWEET11 and AtSWEET12 transporters function in tandem to modulate sugar flux in Arabidopsis. The present study explored the homology models of AtSWEET11 and AtSWEET12 and revealed the key amino acids essential for substrate recognition and transport.
Docking studies showed that the central sucrose binding residues are almost similar in both AtSWEET11 and AtSWEET12 transporters. We deduced that the structural similarities between AtSWEET11 and AtS-WEET12 is responsible for the functional redundancy of these two transporters. However, it is interesting to note that the CTD varies between AtSWEET11 and AtSWEET12 and their orthologs in different plant species. It is highly plausible that these transporters are independently regulated through PTM at the CTD, which might be a reason for distinct and exclusive roles of these two transporters in various plant physiological processes. The differential expression of Anjali for critical reading of the manuscript.